Abstract

Background

Congenital long-QT syndrome (LQTS) may present during fetal development and can be life-threatening. The molecular mechanism for the unusual early onset of LQTS during fetal development is unknown.

Objective

We sought to elucidate the molecular basis for severe fetal LQTS presenting at 19-weeks gestation, the earliest known presentation of this disease.

Methods

Fetal magnetocardiography was used to demonstrated torsade de pointes and a prolonged rate-corrected QT interval. In vitro electrophysiological studies were performed to determine functional consequences of a novel SCN5A mutation found in the fetus.

Results

The fetus presented with episodes of ventricular ectopy progressing to incessant ventricular tachycardia and hydrops fetalis. Genetic analysis disclosed a novel, de novo heterozygous mutation in SCN5A (L409P) and a homozygous common variant (R558). In vitro electrophysiological studies demonstrated that the mutation in combination with R558 caused significant depolarized shifts in voltage-dependence of inactivation and activation, faster recovery from inactivation and a 7-fold greater level of persistent current. When the mutation was engineered in a fetal-expressed SCN5A splice isoform, channel dysfunction was markedly potentiated. Also, R558 alone in the fetal splice isoform evoked a large persistent current, hence both alleles were dysfunctional.

Conclusion

We report the earliest confirmed diagnosis of symptomatic LQTS, and present evidence that mutant cardiac sodium channel dysfunction is potentiated by a developmentally regulated alternative splicing event in SCN5A. Our findings provide a plausible mechanism for the unusual severity and early onset of cardiac arrhythmia in fetal LQTS.

Introduction

Congenital long-QT syndrome (LQTS) refers to a group of disorders with primary impairment of myocardial repolarization predisposing to life-threatening cardiac arrhythmias especially torsade de pointes (TdP) that are caused by genetic mutations in cardiac ion channels or channel modulating proteins.1 The disease is typically recognized in late childhood or early adolescence but extreme cases may present during infancy or in the perinatal period.2-5 Clinical signs suggestive of fetal LQTS include ventricular tachycardia, second degree atrioventricular (AV) block and, most commonly, sinus bradycardia,6, 7 but such findings may go undetected owing to the lack of routine electrocardiographic testing of fetuses. Evidence for Mendelian inheritance is not always apparent in cases of fetal LQTS because of de novo mutations or germ line mosaicism.8, 9 Certain SCN5A mutations, many of which are de novo,2, 4, 5, 10-14 present with earlier onset and more severe congenital arrhythmia syndromes than is typical for LQTS. The reason for greater severity and lethality of certain genetic variants during early life is unknown.

Here we report the clinical, electrocardiographic and genetic diagnosis of LQTS in a fetus at 19 weeks gestation presenting with ventricular tachycardia and severe hydrops fetalis. To our knowledge, this is the earliest gestational age at which a diagnosis of LQTS has been made after being suspected on the basis of clinical presentation. A novel, de novo SCN5A mutation combined with a common genetic variant was discovered in the proband and we demonstrated a plausible molecular basis for arrhythmia presentation. Specifically, we determined that the mutation and common variant both conferred severe functional disturbances when expressed in the context of a cardiac sodium channel isoform generated by a developmentally regulated SCN5A alternative splicing event. Our findings implicate dysfunction of a fetal-expressed sodium channel splice isoform as a predisposition to intrauterine mortality in LQTS. These results may have relevance to perinatal and neonatal death in other clinical settings.

Testing for mosaicism

Measurement of cardiac SCN5A expression

De-identified, frozen, postmortem heart tissues from white-American subjects were obtained from the Brain and Tissue Bank of the University of Maryland under an exemption for human subject research granted by the Vanderbilt University institutional review board. Total RNA was isolated and used for real-time quantitative RT-PCR using TaqMan probes specific for SCN5A exon 6 or 6A to measure relative levels of mRNA transcripts containing either of these alternate exons. Additional details of these experimental methods are provided in the online supplement.

Mutatgenesis of recombinant human cardiac sodium channel (NaV1.5) was performed as described previously5, 15 except that a rare variant present in the original cDNA (glutamine-1027; Genbank accession number M77235)16 was reverted to the common allele (arginine-1027). The common variant R558 was engineered in some constructs to match the genotype of the study subject. Wild-type (WT) or L409P mutant channel cDNA (0.5 μg) were transiently transfected into tsA201 cells using FuGene 6 (Roche Diagnostics, Indianapolis, IN) combined with a plasmid encoding enhanced green fluorescent protein (IRES2-eGFP, 0.5 μg). Transiently transfected cells were incubated 48 hours at 37°C prior to electrophysiological measurements. Cells exhibiting green fluorescence were selected for patch-clamp recordings. A fetal NaV1.5 cDNA was engineered by making the following amino acid substitutions encoded by the alternate exon 6 (designated exon 6A): V206T, S207T, N209F, I210V, K211D, L215V, P234S.

In vitro electrophysiology

Sodium currents were recorded at room temperature using the whole-cell patch clamp technique as described previously5, 15 with additional details provided in the online supplement. Results are presented as mean ± SEM. Unless otherwise noted, statistical comparisons were made by using an unpaired Student t test in reference to WT NaV1.5. Statistical significance was assumed for P < 0.05.

Results

Identification of a novel SCN5A mutation in a fetus with torsade de pointes

A 29-year-old primiparous woman was referred for evaluation of an irregular fetal heart rhythm at 19
weeks gestation (by last menstrual period and an 11 week ultrasound). There was no family history of pregnancy loss, syncope, seizures, sudden cardiac death at any age, accidental death or drowning. An initial fetal echocardiogram at 20 weeks gestation disclosed normal cardiac anatomy with decreased ventricular function, mild tricuspid valve insufficiency and a very small pericardial effusion. The atrial rate was regular at 130-160 beats per minute (bpm) but the ventricular rate was variable. There were frequent premature ventricular contractions and couplets, and short (3-4 beat) runs of tachycardia (Fig. 1A). During non-sustained tachycardia, the atrial rate was slower than the ventricular rate, leading to a presumptive diagnosis of ventricular tachycardia. There was no evidence of AV block. Maternal electrolytes were normal, and serum testing for maternal SSA/SSB antibodies, IgG to cytomegalovirus and Toxoplasma gondii were negative. The QTc intervals determined from 12-lead ECG recordings were in the normal range for the patient and fetus' father (424 ms and 383 ms, respectively). At 20
weeks, a fetal magnetocardiogram (fMCG) revealed frequent short episodes of polymorphic ventricular tachycardia consistent with TdP and a QTc interval of 604 msec (Fig. 1B & 1C). During 2 hours of data recording, AV block was not observed. An ultrasound the same day showed interval accumulation of pleural fluid and ascites consistent with hydrops fetalis. Treatment of the fetal arrhythmia was discussed with the family, however, because of the dire clinical status, the parents elected not to pursue treatment.

Echocardiography at 22 weeks gestation revealed severe cardiac dysfunction, more frequent and more prolonged episodes of ventricular tachycardia (Fig. 1D), and worsening hydrops fetalis. Although tachycardia cycle length was similar to that observed 2 weeks prior (Fig. 1A), the velocity of the Doppler signals were extremely low, suggesting that the stroke volume was greatly decreased during tachycardia episodes. At this time, tachycardia episodes had a longer duration, and the intervals between tachycardia episodes were shorter (not shown) implicating increased tachycardia burden as a factor for the progression of cardiac dysfunction. Based on the extent of clinical deterioration, pregnancy was terminated at the request of the family. Postmortem genetic testing (Familion®) of the fetus identified a novel heterozygous SCN5A transition mutation (T1226C) predicting a missense change in codon 409 from leucine to proline (designated SCN5A-L409P). No mutations were identified in ten other LQTS genes interrogated by the commercial genetic test (KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9, SNTA1). The fetal proband was also homozygous for a common nonsynonymous variant, R558 (rs1805124). However, analysis of parental DNA identified no evidence of germline mosaicism suggesting that SCN5A-L409P occurred on a de novo basis in the fetus.

Functional Consequences of SCN5A-L409P/R558

We determined the functional consequences of the mutation by in vitro electrophysiological recording of heterologously expressed recombinant human cardiac sodium channels (NaV1.5). To recapitulate the genotype of the fetus, we engineered the L409P mutation along with the R558 variant (combination allele designated as L409P/R558). Expression of mutant channels in tsA201 cells generated voltage-dependent sodium currents that exhibited several differences compared to wildtype (WT) NaV1.5 including a trend toward reduced peak current density (Fig. 2A & B), significant depolarized shifts in the voltage-dependence of steady-state inactivation and conductance-voltage relationships (Fig. 2C; Supplemental Table S1) and markedly accelerated recovery from inactivation (Fig. 2D). Additionally, as compared to WT channels, L409P/R558 exhibited a 7-fold greater level of persistent sodium current measured as a percentage of peak current (1.4 ± 0.2%, Fig. 3A; Table 1). Increased persistent current is the most frequently observed functional disturbance exhibited by SCN5A mutations associated with LQTS,17 and can also explain abnormal inward current evoked by a slow depolarizing voltage ramp (Fig. 3B & C). These findings indicate that L409P/R558 causes dysfunction of cardiac sodium channels consistent with cardiac arrhythmia predisposition. However, these findings do not provide an explanation for the severe intrauterine presentation of ventricular arrhythmia.

Developmental regulation of SCN5A exon 6 splicing

We hypothesized that the severity of LQTS presentation in this fetus was due to more severe functional consequences conferred by the mutant genotype in the background of a fetal-expressed alternatively spliced SCN5A transcript. We focused on the developmentally timed alternative splicing of exon 6 that generates two splice isoforms differing by seven amino acid residues within a voltage-sensing domain (D3/S3-S4) originally described in neuroblastoma cells.18 We determined the relative levels of SCN5A mRNA transcripts containing the canonical exon 6 or the alternative exon 6A in human heart samples representing various developmental stages including the fetal period (28-33 weeks gestation), infancy (2-6 months of age) and adulthood (> 18 years of age).

In fetal human heart, we observed ~1.5-fold greater levels of SCN5A mRNA transcript containing exon 6A, encoding what we designated as fetal NaV1.5, as compared with transcripts containing the canonical exon 6, encoding the adult splice variant of NaV1.5 (Fig. 4). In hearts from infants, the relative abundance of adult and fetal NaV1.5 mRNA was approximately equal, while in adult heart there was a 7.5-fold higher level of adult NaV1.5 mRNA as compared to the fetal splice variant. The fetal (exon 6A) to adult (exon 6) NaV1.5 expression ratio in adult heart was significantly different from both infant and fetal heart (p<0.0001) whereas differences between fetal and infant heart expression ratios were not significant (p=0.06). We conclude that SCN5A exhibits a developmental switch in exon 6/6A alternative splicing in human heart during early postnatal life.

Functional properties of WT and mutant fetal NaV1.5

To test whether the functional consequences of L409P/R558 are different in fetal NaV1.5, we engineered a recombinant human fetal NaV1.5 cDNA and compared its electrophysiological properties to adult NaV1.5. We demonstrated that fetal NaV1.5 exhibits a significantly more positive midpoint (+9 mV shift in V1/2) in the conductance-voltage relationship as compared with the adult splice variant, but no significant differences in peak current density, voltage-dependence of steady-state inactivation curve or recovery from inactivation (Supplemental Figure S1; Table S1). These data demonstrate intrinsic differences in biophysical properties between human fetal and adult NaV1.5 splice isoforms.

We investigated the consequences of the common SCN5A variant R558 on the functional properties of fetal NaV1.5 and compared these to the effect of the variant on the adult splice isoform. Whereas, R558 has minimal functional impact on adult NaV1.5 (Supplemental Table S1), the expression of this variant in fetal NaV1.5 demonstrates substantial effects including lower whole cell current density and a large persistent sodium current (Fig 5; Table 1). These findings demonstrate a considerable functional defect for the non-mutant allele.

Finally, we determined the functional consequences of L409P/R558 in fetal NaV1.5. Compared with biophysical properties of this mutation in the adult isoform, L409P/R558 in fetal NaV1.5 exhibited greater depolarizing shifts in steady-state inactivation and in conductance-voltage relationships (Fig. 6 and Supplemental Table S1), and greater persistent current (Fig 6; Table 1). Superimposed, normalized current traces (Fig. 6D) and quantitative analysis also illustrate slower activation rise time and slower kinetics of inactivation for fetal NaV1.5-L409P/R558 as compared with WT fetal NaV1.5, functional defects that were not as prominent in the adult splice isoform (Supplemental Fig S2). Collectively, these observations indicate that both SCN5A alleles carried by this fetus, one with R558 alone and the other with L409P/R558, cause much more profound sodium channel dysfunction in the background of fetal NaV1.5 splice isoform providing a plausible explanation for severe presentation of intrauterine LQTS.

Discussion

We report, to our knowledge, the earliest confirmed diagnosis of symptomatic LQTS in a 19-week fetus. We also demonstrated a plausible mechanism for the early onset and unusual severity of the condition that involves interaction of the causative SCN5A mutation with the product of a developmentally regulated alternative splicing event in this gene. Our findings help explain how genetic cardiac arrhythmia susceptibility including LQTS can contribute to fetal mortality.6, 19-21

Clinical and genetic features of fetal LQTS

The phenotype we described in a second trimester fetus associated with a previously undocumented de novo SCN5A mutation included ventricular ectopy and occasional periods of ventricular tachycardia, followed by a rapid progression to sustained episodes of TdP, impaired ventricular systolic function and severe hydrops fetalis. The diagnosis of LQTS in utero was facilitated by using fMCG and confirmed post-mortem by genetic testing. This unusually severe, early onset fetal arrhythmia was without other features typical of intrauterine LQTS presentations. The most common early manifestation of fetal LQTS is sinus bradycardia, which has been documented in some cases at less than 25 weeks gestation, whereas other rhythm disturbances associated with fetal LQTS including second-degree AV block and TdP are most typical between 28 and 40 weeks gestation.22-24

A fetal diagnosis of ventricular tachycardia should prompt an evaluation for LQTS. In a review of recent literature regarding ventricular tachycardia in the fetus, 9 of 22 cases were confirmed to have LQTS.6, 9, 23, 25, 26 Moreover, of the LQTS cases, only 4 survived the neonatal period, suggesting that fetuses presenting with ventricular tachycardia, especially TdP, due to LQTS have a particularly poor prognosis. Fetal LQTS should also be considered in the differential diagnosis of complex fetal arrhythmia with fetal hydrops or unexplained fetal loss even in the absence of a family history. While the prevalence of fetal demise with LQTS is unknown, it is noteworthy that 30% of stillbirths are unexplained27 and de novo LQTS gene mutations may account for a portion of these cases.

Carriers of certain SCN5A mutations, many of which are de novo, may present with earlier onset and more severe congenital arrhythmia syndromes.2, 4, 5, 10-14 Maternal mosaicism for SCN5A mutation has also been described in association with recurrent third-trimester fetal hydrops or stillbirth.8 Interestingly, SCN5A mutations are present in the preponderance of reported fetal and perinatal LQTS cases whereas only ~10% of LQTS in older children and young adults is explained by this genotype. The high rate of reported de novo mutations in the perinatal period may reflect low heritability due to a survival disadvantage conferred by severe phenotypes. Our report taken together with previous literature suggests a diagnosis of LQTS, with particular suspicion for SCN5A mutations, should be entertained in a fetus with ventricular arrhythmia even in the absence of a family history or parental genetic diagnosis of LQTS.

Alternative SCN5A splicing potentiates LQTS mutation severity

The novel SCN5A mutation we identified in this study encodes a dysfunctional cardiac sodium channel, but the degree of channel dysfunction determined in the context of the canonical adult NaV1.5 splice product did not explain the unusual severity and early onset of fetal arrhythmia. We considered that using the adult NaV1.5 splice isoform to evaluate the functional consequences of a mutation associated with an intrauterine arrhythmia might be misleading if another molecular form of the channel was predominant in fetal heart. SCN5A undergoes alternative mRNA splicing to generate multiple isoforms of the protein.28 Although many described splicing events have uncertain physiological significance, at least one major alternative splicing event could have implications for understanding severe fetal LQTS. Specifically, a developmentally regulated SCN5A splicing event involving selection between two alternative forms of exon 6 generates NaV1.5 isoforms that differ at several amino acid residues within a voltage-sensor domain (D1/S3-S4).18, 28 This alternative NaV1.5 splice variant is strongly expressed in neonatal mouse heart but is down-regulated later in development.29 Evidence for developmentally regulated expression in humans was not previously been demonstrated. Here we show prominent expression of alternatively spliced NaV1.5 mRNA incorporating exon 6A in fetal and infant heart. Previous studies have referred to this product of alternative splicing as a ‘neonatal’ SCN5A, but given the high level of expression we observed in fetal human heart (Fig. 4), we suggest that fetal NaV1.5 is a more appropriate designation.

Hence, we investigated the functional consequences of the mutation in the fetal NaV1.5 splice isoform. These experiments indicated that the common variant (R558) and the compound mutant genotype (L409P/R558), the two alleles carried by the proband, each conferred much greater functional defects on fetal NaV1.5 than on adult NaV1.5. In particular, the proportion of persistent current was substantial for channels with L409P/R558 (11% of peak current) as well as with the R558 common variant alone (3.7% of peak current, Table 1), and was also abnormal for mutant adult NaV1.5. Therefore, this fetus expressed mostly dysfunctional SCN5A alleles at the time of the presenting arrhythmia syndrome. The profound defects observed for mutant fetal NaV1.5 channels help explain both the malignant character of the arrhythmia syndrome and the early onset of this condition in utero. The dysfunction of fetal NaV1.5-R558 suggests that carriers of this common allele may have greater risk for fetal or perinatal arrhythmias, but this seems incongruous with genotype frequencies observed in the general adult population (http://www.ncbi.nlm.nih.gov/snp/?term=rs1805124). We speculate that R558 alone is not sufficient to evoke arrhythmia risk in the fetus but may act as a modifier of other variants as previously demonstrated for other SCN5A mutations having later clinical presentations.30-33 We further speculate that impaired ventricular contractility was also the result of severe sodium channel dysfunction and related to either tachycardia as suggested by fetal Doppler studies (Fig. 1D) or disturbances in intracellular ionic homeostasis proposed for familial dilated cardiomyopathy associated with SCN5A variants.34

Conclusion

In summary, we present a case of fetal LQTS identified early in mid-gestation due to a novel, de novo SCN5A mutation. Fetal LQTS may be an under-recognized cause of early fetal hydrops and unexplained fetal loss, and should be considered in the differential diagnosis of the fetus with rapidly progressive heart failure and complex arrhythmia even if family history is negative. The unusual severity and early onset of arrhythmia in this case were explained by profound dysfunction of the mutant sodium channel carried by the fetus that was revealed only by considering the correct molecular context, a fetal-expressed alternatively spliced SCN5A transcript. Our findings demonstrate an important contribution of developmentally regulated alternative SCN5A splicing to the genetic risk for prenatal life-threatening cardiac arrhythmia.

Supplementary Material

01

Acknowledgments

Funding Sources: This work was supported by grants from the National Institutes of Health (HL083374 to A.L.G.; HL063174 to R.T.W.; and HL69712 to D. W. B.).

Abbreviations

LQTS

long-QT syndrome

fMCG

fetal magnetocardiography

AV

atrioventricular

WT

wild type

TdP

torsade de pointes

Footnotes

Conflicts of Interest: None

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